Process for the selective alkylation of-sh groups in proteins and peptides for the study of complex protein mixtures

ABSTRACT

Weakly basic molecules containing a double bond (in particular vinylpyridines) are able to react and selectively alkylate —SH groups in proteins, thus preventing their re-oxidation to disulphur bridges. Contrary to conventional alkylating agents, such as iodoacetamide, such molecules reach 100% alkylation of all —SH residues, even in complex proteins, without reacting with other functional groups. Their use is particularly effective in proteome analysis and more generally for analysing proteins in which the —SH groups should be blocked. Additionally, the use of vinylpyridines partially or totally deuterated, and thus with a mass difference as compared to non-deuterated vinylpyridines, allows studies of induction/repression of protein synthesis.

DESCRIPTION OF THE INVENTION

The present invention refers to a process for the selective andefficient alkylation of —SH groups in proteins. The process of theinvention is useful in a number of analytical techniques for proteinanalysis and characterization.

BACKGROUND OF THE INVENTION

In the post-genomic era an emerging field is the science called“proteomics”, i.e. a research area with the aim of the global analysisof gene expression, via a combination of methods for resolving,identifying, quantifying and characterizing all proteins present in atissue, in a cellular lysate, in a biological fluid, in an entireorganism (Wilkins, M. R., Williams, K. L., Appel, R. D., Hochstrasser,D. F., Eds., Proteome Research: New Frontiers in Functional Genomics,Springer, Berlin, 1997). The term “proteome” is a newly introduced word,which signifies precisely the entire set of proteins expressed by thegenome. Since the proteome, even of a simple cell lysate, could beextremely complex (comprising several thousands of proteins), for itsanalysis one has adopted powerful separation methods calledtwo-dimensional (2-D) maps, which couple a charge-based separationmethod (isoelectric focusing, IEF), in the first dimension, to amass-based separation method (SDS-PAGE, polyacrylamide gelelectrophoresis in presence of sodium dodecyl sulphate) in the seconddimension. This method, introduced already in 1975 (O'Farrell, P. H., J.Biol. Chem. 250, 1975, 4007-4021), has been refined over the years withthe advent of immobilized pH gradients (Righetti, P. G., Immobilized pHGradients: Theory and Methodology. Elsevier, Amsterdam, 1990), of newsurfactants (Chevallet, M. et al., T., Electrophoresis 19, 1998,1901-1909), of new staining methods (Rabilloud, T., Anal. Chem. 72,2000, 48A-55A).

The main problems in the analysis of complex proteins mixtures include:(a) the complete sample solubilization; (b) the elimination of artifactsduring the various electrophoretic steps. In order to solve the firstproblem, new surfactants, of the amido-sulphobetaine type, incombination with chaotropic agents, such as urea/thiourea (Rabilloud, T.et al., J., Electrophoresis 18, 1997, 307-316), have been described. Asfor the elimination of artefacts, it has been recently demonstrated thatthe reduction and alkylation of disulfide bridges is a fundamental stepfor preventing the formation of spurious protein zones in the 2-D map.The sole reduction of disulfide bridges in naive proteins (typicallyobtained with an excess of 2-mercaptoethanol of dithiothreitol) cannotavoid artefacts, especially in the first IEF dimension. In fact, duringthe IEF separation, especially in an alkaline milieu, the reduced —SHgroups spontaneously re-oxidize, generating artefactual bands, due tohomo- and hetero-oligomers among the same or different polypeptidechains (Herbert, B. et al., Electrophoresis 22, 2001, 2046-2057). Thus,it has been proposed to reduce and alkylate proteins as a fundamentalpre-treatment of the sample, prior to any separation step. The standardalkylating agent has always been iodoacetamide, recommended since thefifties by protein chemists and universally adopted in all protocols ofanalysis. Recent data, though, have demonstrated severe limitations ofthis alkylating agent:

-   -   (a) to start with, it is impossible to obtain 100% alkylation of        all reduced —SH groups (Galvani, M. et al., Electrophoresis 22,        2001, 2058-2065), thus leaving intact a number of free —SH        groups, potentially able to generate spurious bands. If the        alkylation process is prolonged for long times (>6 hours), not        only the reaction of free —SH groups does not progress, but        non-selective alkylation of other reacting groups takes place,        in particular of ε-amino groups of Lys;    -   (b) additionally, the alkylation reaction is strongly inhibited        by the presence of numerous surfactants adopted for solubilizing        complex protein mixtures;    -   (c) finally, it has been recently reported that iodoacetamide        reacts rapidly with one of the solubilizing agents universally        adopted until now in protein analysis, and precisely with        thiourea. The addition of iodoacetamide to thiourea (present in        a strong excess) is even more rapid than the addition to the —SH        groups in proteins, a parasitic reaction which strongly quenches        the reaction yield (Galvani, M. et al., Electrophoresis 22,        2001, 2066-2074).

DESCRIPTION OF THE INVENTION

It has now been found a process which overcomes all the above drawbacks.

The process of the invention is based on the finding that weakly basiccompounds having double bonds react with the reduced —SH groups ofproteins quantitatively and selectively (i.e. by avoiding side reactionwith other protein groups, such as lysines, tyrosines, terminal —NH₂groups). Moreover, said weakly basic compounds do not give spuriousreactions with other components of the solubilizing mixture, such asthiourea and are not inhibited by the surfactants typically utilized insaid solubilizing mixtures.

The weakly basic compounds useful in the process of the inventionpossess the following structural characteristics:

-   -   (a) the presence of one or more weakly basic nitrogen groups;    -   (b) the presence of an acrylic-type double bond.

The present invention accordingly provides a process for the selectivealkylation of —SH groups in a protein, comprising the reaction inneutral or alkaline milieu of said protein with a weakly basic compoundhaving:

-   -   (a) one or more weakly basic nitrogen groups;    -   (b) at least one acrylic-type double bond,    -   wherein the percent alkylation of the total —SH groups is higher        than 90%.

The invention further concerns a method for the analysis of proteins bymeans of electrophoretic or analytical techniques, comprising apreliminary alkylation of the —SH groups of the proteins to be analyzedby the process as defined in the claims.

DESCRIPTION OF THE FIGURES FIG. 1

Alkylation kinetics of reduced bovine α-lactalbumin after incubationwith 100 mM IAA at pH 9.0. The time points have been taken up to 24hours. Panel a: control (m/z=14191); panel b: 2 min of incubation (them/z 14653 represents the octa-alkylated peak); panel c: 2 hours ofincubation; panel d: 24 hours of reaction time (the m/z 14707 peakrepresent the nona-alkylated species; the desired product, m/z=14652 isnow the minor components among a highly heterogeneous series ofproducts). Analysis by mass spectrometry in the MALDI-TOF mode.

FIG. 2

MALDI-TOF mass spectra of bovine α-lactalbumin after 1 h incubation withDMA (panel a) or 4-vinylpyridine (panel b), both in presence of thesurfactant 2% Triton X-100. Note that, in panel b, the peak at m/z 15248represents an adduct of LCA with the MALDI matrix, sinapinic acid.

FIG. 3

MALDI-TOF mass spectra of bovine α-lactalbumin after 1 h incubation with2% amidosulphobetaine-14 and: (a) acrylamide, (b) 2-vinylpyridine and(c) 4-vinylpyridine. In all cases the reaction has been carried out atpH 7.0. Note, in both panels b and c, a single reaction channel of LCAwith 2VP and 4VP, respectively. The higher order peaks (m/z 15248 and15468 in b and c) represent adducts LCA with the MALDI matrix, sinapinicacid.

FIG. 4

Reaction of 4-vinylpyridinev(panels a and b) and of acrylamide (panel c)with the —SH groups of lysozyme. In (a) the reaction has been carriedout at pH 9.0, whereas at pH 7.0 in panels (b) and (c).

FIG. 5

Isotopic ratio in the peptides of a1-acid glycoprotein (from rat sera)labelled with either light or heavy (hepta-deuterated) 2-vinylpyridine.After labelling, the two rat sera were mixed in a 70:30 (heavy/light)ratio and fractionated via two-dimensional maps. The zone of the a1-acidglycoprotein (M_(r) 21546, pI 5.0) was eluted, digested with trypsin andanalysed by mass spectrometry in the MALDI-TOF mode. The insert showstwo fragments at m/z 1235.0 and 1241.9. The difference in m/z of 6.9coincides with the difference between the 2-d₇-VP and the non-deuterated2-VP. This observation, together with the ratio of the relativeintensities of the two signals (ca. 68:32) confirms that this peptidecontains a single Cys residue which reflects the ratio of the twoisotopic markers (70:30) in the original protein mixture.

DETAILED DESCRIPTION OF THE INVENTION

Examples of preferred weakly basic compounds which may be used in theprocess of the invention are vinylpyridines, such as 2- and4-vinylpyridine, having the following formulas:

3-vinylpyridine may also be used, even though precaution should be usedin view of its higher propensity to auto-polymerization.

The use of a vinylpyridine as alkylating agent for —SH groups inproteins has been only generically mentioned by M. J. Dunn (in: GelElectrophoresis: Proteins, Bio. Sci. Publ. Oxford, 1993). Nevertheless,vinylpyridines have not been considered in the actual laboratorypractice, the characteristics of extreme selectivity, completereactivity and very high specificity of such molecules having not beendisclosed.

The process according to the present invention allows for the alkylationof more than 90% of the —SH groups of a given protein, preferably morethan 95% and even more preferably about 100%.

The process of the invention moreover can be utilized for proteinalkylation both at alkaline and neutral pH values; it is compatible withthe presence in the protein sample of surfactants, of zwitterionic(e.g., CHAPS, amidosulphobetaines), of neutral (Triton X-100, NonidetP-40) of anionic (e.g., SDS) type. Vinylpyridines are additionally fullycompatible with the chaotropic agents usually adopted for solubilizingcomplex protein mixtures, such as urea and thiourea, since they do notreact with these compounds under the usual experimental conditions.

The protein, after reduction with a suitable reducing agent such asdithiotreitol, is reacted with the weakly basic compounds according tothe invention at a temperature ranging from about 15 to about 30° C.,usually, from about 20 to about 25° C., for a period of time from 30′ toabout 6 hours, usually from about 45′ to 2 hours. The reaction solventis not critical and it generally consists of an aqueous buffer such as aphosphate or a Tris-HCl buffer. An excess of the weakly basic compoundwill be generally used, for instance 100 mM for a 50 μM solution of theprotein to be analyzed.

The vinylpyridines can optionally be deuterated, either totally(hepta-deuterated) or partially (in correspondence to the vinyl moietyor the pyridine ring), for the quantitative studies of protein synthesisin a biological sample, according to the method disclosed in Gygi, S.P., Aebersold, R., in Proteomics: a Trend Guide, Blackstock, W., Mann,M., eds., Elsevier, London, 2000, pp. 31-36).

The process of the invention is also advantageously applied in a numberof analytical methods and particularly in:

-   -   proteome analysis or in the analysis of complex protein/peptide        mixtures, either under naive or denatured conditions;    -   electrophoretic two-dimensional maps, said maps comprising a        first dimension by isoelectric focusing (either conventional or        in immobilized pH gradients) followed by a second SDS-PAGE        dimension.    -   electrophoretic methods, either mono-, bi- or multi-dimensional,        either in a free phase or on various gel types (including, but        not limited to, polyacrylamide and agarose gels);    -   capillary electrophoresis, either in free phase or in sieving        liquid polymers, in presence of SDS, in the isoelectric focusing        mode, in conventional capillaries, in micro- and nano-chips;    -   a combination of the above methods with other methods, such as        blotting and mass spectrometry, either on-line or off-line;    -   chromatographic separations, either mono-, bi or        multi-dimensional;    -   mixed-type separations, electrophoretic/chromatographic, either        bi- or multi-dimensional;    -   pre-fractionation procedures in proteome analysis, or in general        in analysis of complex protein/peptide mixtures, via        electrophoretic or

The invention is illustrated in more detail in the following Examples,in comparison to iodoacetamide and to neutral derivatives of acrylamide,including N-substituted acrylamides.

Comparative Example No. 1

Alkylation kinetics of bovine α-lactalbumin (LCA) with iodoacetamide(IAA). A 50 mM solution of LCA in 50 μM Tris-acetate, pH 9.0 and 7 Murea, is reduced with 50 mM dithiothreitol (DTT) for 1 hour at 25° C.After reduction, the protein is incubated with 100 mM IAA and thealkylation kinetics followed up to 24 hours via mass spectrometry in theMALDI-TOF (matrix-assisted, laser desorption ionization—time of flight)mode. LCA contains, in the polypeptide chain, 8 cysteine residues, andtherefore 8 —SH groups, which could potentially undergo alkylation. Asshown in FIG. 1, already after 2 min the octa-alkylated derivativesrepresents only the 60% of all reaction products (panel b, peak at m/z14653), the remaining 40% representing, hepta-, hexa- andpenta-alkylated species, in this order. However, one can notice that,after 2 hours of reaction, the octa-alkylated derivative is barelyincreased to 70% (panel c, peak at m/z 14656), the remaining 30% beingrepresented by derivatives of lower degree of alkylation. The reactiondoes not seem to progress any further for longer incubation times: after24 hours, other groups present in the protein are alkylated instead(especially the ε-amino groups of lysine) (panel d; e.g., the peak atm/z 14707 is a nona-alkylated derivative, and represents the main peak;in addition one can notice peaks which could be deca-substituted andeven at a higher degree of substitution; the final reaction product, inany event, is strongly heterogeneous and the desired product, at m/z14652, represents now the minor component). The lack of quantitativereaction and the number of side reactions, on other amino acid residuesare strongly hampering a proper proteome analysis, since they make thecorrect identification of proteins difficult, presently mostly performedvia mass spectrometric analysis. In addition, the extra reactions onlysines and other residues originate spurious spots in 2-D maps, in thatthey generate new protein isoforms having a different isoelectric point(pI).

Example No. 2

Alkylation of bovine α-lactalbumin (LCA) with dimethyl acrylamide (DMA)and 4-vinylpyridine in presence of neutral surfactants. A 50 ,μMsolution of LCA in 50 mM Tris-acetate, pH 9.0 and 7 M urea, is reducedwith 50 mM dithiothreitol (DTT) for 1 hour at 25° C. After reduction,the protein is incubated with 100 mM of either DMA or of 4-vinylpyridine(4VP) for 1 hour at 25° C. Both reactions are carried out in presence of1% Triton X-100, one of the possible surfactants adopted in proteomeanalysis. The reaction products are monitored via mass spectrometry inthe MALDI-TOF mode. As shown in FIG. 2, whereas in the case of 4VP onlyone reaction product is present (m/z 15083), corresponding to the LCAadduct with 8 4VP residues, in the case of DMA there are still someamounts of unreacted product (m/z 14213) accompanied by a whole seriesof reaction species, the most abundant components of which are tri- andtetra-alkylated species. 4VP turns out to be by far the most efficientreagent, able to carry out the reaction to full completion, while beingunaffected by the inhibitory effect of surfactants.

Example No. 3

Alkylation of bovine α-lactalbumin (LCA) with acrylamide and with 2-or4-vinylpyridine in presence of zwitterionic surfactants at neutral pH.Protein alkylation is typically carried out at pH values between 8.5 and9.0, a pH which facilitates the reaction between the —S⁻ group (pK 8.3),negatively charged, and neutral agents such as IAA and acrylamide. Atthis pH value, though, and for long reaction times, spurious reactiontake place, such as alkylation of the ε-amino groups of lysine. On thecontrary, the 2- and 4-vinyl pyridines are weak bases (pKs ca. 5.2 to5.6), which, at pH 5, exist in a protonated form, thus rendering themhighly reactive with deprotonated —SH groups. Thus, an interestingreaction pH could be at pH around neutrality, i.e. half a way in betweenthe pK values of the two reacting species. At pH ca. 7 both thealkylating agent and the —SH group would carry a partial positive andnegative charge, respectively, which would render them highly reactivetowards each other. A 50 μM solution of LCA in 50 mM phosphate buffer,pH 7.0 and 7 M urea, was reduced with 50 mM dithiothreitol (DTT) for 1hour at 25° C. After reduction, the protein was incubated with 100 mM ofeither acrylamide or of 2- (2VP) or 4- (4VP) vinylpyridine for 1 hour at25° C. Both reactions were carried out in presence of 1%amidosulphobetaine (ASB), a zwitterionic surfactants much in vogue todayin proteome analysis. The reaction products were monitored via massspectrometry in the MALDI-TOF mode. As shown in FIG. 3, whereas in thecases of 2VP and 4VP (panels b and c) only one reaction product ispresent at m/z 15038, corresponding to the adduct of LCA with 8 residuesof 2VP or 4VP, in the case of acrylamide there still remains asubstantial amount of unreacted product, accompanied by a series ofreaction products, of which the most abundant species are tri- andtetra-alkylated compounds. This clearly shows how 2VP and 4VP representby far the most efficient reagents, able to carry out the reaction tofull completion, while being unaffected by the inhibitory effect ofsurfactants.

Example No. 4

Alkylation of lysozyme with acrylamide or 4-vinylpyridine at alkalineand neutral pH values. We have also investigated whether the isoelectricpoint (pI) of the protein could influence the —SH group alkylationreaction. This could be a drawback, particularly in the case of 2VP and4VP (due to their weakly basic characteristics), in that the excess ofpositive charges on such alkaline proteins could inhibit their access tothe reaction sites. We have thus studied the behaviour of chicken egglysozyme, a protein with a pI of ca. 10, also containing 8 cysteineresidues in the polypeptide chain. A 50 μM solution of lysozyme in 50 mMphosphate buffer, pH 7.0, or in Tris-acetate buffer, pH 9.0, both inpresence of 7 M urea, was reduced with 50 mM dithiothreitol (DTT) for 1hour at 25° C. After reduction, the protein was incubated with 100 mM ofeither acrylamide or of 4VP for 1 hour at 25° C. The reaction productswere monitored via mass spectrometry in the MALDI-TOF mode. As shown inFIG. 4, in the case of 4VP (panel a for the pH 9.0, panel b for the pH7.0 reactions) only a single reaction product is present (m/z 15164),corresponding to the adduct of lysozyme with 8 residues of 4VP, whereas,in the case of acrylamide, the main peak is the m/z 14893 (correspondingto the adduct with 8 acrylamide residues), but this peak is contaminatedby sizeable amounts of m/z 14750 and m/z 14822, corresponding to thehexa- and hepta-alkylated products, respectively. Here again, it is veryclear how 4VP (and its analogue 2VP) are able to alkylate to 100% alsobasic proteins at unfavourable pH values, such as pH 7.0, a pH at whichthe positive charge of lysozyme would be considerably increased ascompared to pH 9.0.

Example No. 5

Alkylation of proteins having large Mr values, with a high cysteinecontent, with IAA or 2VP or 4VP at neutral pH. In the above examples, wehave followed the alkylation of small-size proteins (13-15000 Da) andwith an average content of —SH groups (8 Cys residues). It was ofinterest to follow the same reaction on medium to high mass proteins andwith a higher content of Cys residues. Two interesting cases arerepresented by human serum albumin (HSA) (M_(r) 66472, pI 5.7,containing 35 Cys residues) and by rabbit phosphorylase (M_(r) 97158, pI6.8, containing 36 Cys residues). Fifty μM solutions of HSA, orphosphorylase, in 50 mM phosphate buffer, pH 7.0 and 7 M urea, werereduced with 50 mM dithiothreitol (DTT) for 1 hour at 25° C. Afterreduction, the proteins were incubated with 100 mM of either acrylamideor of 2- (2VP) or 4- (4VP) vinylpyridine for 1 or 6 hours at 25° C. Thereaction products were monitored via mass spectrometry in the MALDI-TOFmode. The results are reported in Table 1. TABLE 1 Alkylation with 2- o4-VP or with IAA of proteins with medium to high Mr values Total IAA IAA2-VP (4-VP) Protein Mr pI Cys (1 h)* (6 h)* (1 h)* Albumin 66472 5.7 3568% 80% 100% Phosphorylase 97158 6.8 36 65% 78% 100%*The % of alkylation in the tree cases has been measured via massspectrometry in the MALDI-TOF mode of the intact proteins.

Here too one can notice how, in the case of IAA, for both proteins,values of ca. 68% alkylation are reached after one hour of reaction, andbarely of ca. 80% after six hours; conversely, in the case of 4VP (andits analogue 2VP) 100% reaction values are reached after only one hourof incubation.

Example No. 6

Alkylation of proteins with normal or deuterated 2VP or 4VP forquantitative analysis of protein expression. For the study of variationof protein expression in control cells vs. cells treated with a varietyof chemicals (e.g., drugs, inhibitors, promoters, etc.) it is importantto be able to label the two cell populations, separately, with a normal(“light”) or with a deuterated (“heavy”) agent. The two samples are thenmixed, in a 1:1 ratio, and the sample subjected to 2-D map analysis. Theprotein zones which, via analysis with quantitation programs (such asthe PDQuest or Melanie), appear to have varied their expression (byshowing increments or decrements of stain intensity, possibly reflectingup- or down-regulation in protein synthesis) are then eluted from thepolyacrylamide gel and digested with trypsin. The resulting peptides arethen analysed by mass spectrometry in the MALDI-TOF mode. At this point,all peptides marked with 2VP or 4VP are split into two peaks, the lightand heavy ones (this last one marked with the deuterated isotope),spaced apart by 7 Da (in the case of hepta-deuterated pyridines) or by 4Da (in the case in which only the pyridine ring, and not the vinylmoiety, is deuterated) or by 5 Da (in the case of partial deuteration ofthe vinyl moiety). A unit ratio between the areas of the twomono-isotopic peaks means that there has been no variation in proteinsynthesis between the control and treated cells. If, on the contrary,this ratio is higher (or lower) than one, this means that, in thetreated cells, there has been an induction, or repression, of proteinsynthesis. This method thus permits to assess, in a precise andunambiguous manner, which effectors induce or repress protein synthesisand is thus fundamental in evaluating all biological effects induced bydrugs, or the appearance of genetically induced diseases, onset ofcancer etc. The use of isotopic ratios for these quantitative biologicalstudies has already been described in the literature (Gygi, S. P.,Aebersold, R., in Proteomics: a Trend Guide, Blackstock, W., Mann, M.,eds., Elsevier, London, 2000, pp. 31-36), but the alkylating agent is avery complex molecule, the proper reactivity of which has not beendemonstrated, and which is unable of alkylating —SH groups in aquantitative fashion, as in the case of 2VP and 4VP. In addition, theabove agent (called ICAT, isotope coded affinity tag) is biotinylated,since this affinity label is needed for capturing by affinitychromatography only the marked peptides, out of an extremelyheterogeneous peptide population. This is not necessary in the standard2-D map analysis, since only intact proteins are separated, and nottheir peptide digest. We have thus prepared a 2-vinylpyridine eitherpartially or totally deuterated, by using as starting material2-methylpyridine hepta-deuterated, from which one can prepare thecorresponding picolyl-lithium and then, via reaction withparaformaldehyde, obtain the 2-(2-hydroxyethyl)-pyridine, according toJ. Finkelstein et al., Journal of Organic Chemistry 4 (1939) 374-380.From this last compound one can obtain (I. C. Ivanov et al., Arch.Pharm. 322, 1989, 181-190) hepta-deuterated 2-vinylpyridine, when usingd₂-paraformaldehyde, or penta-deuterated, when utilizing non-deuteratedparaformaldehyde, thus with mass differences of 7 and 5 Da as comparedwith “light” (non-deuterated) 2-vinylpyridine. The synthesis of theother two (3VP and 4VP) deuterated vinylpyridines can be obtained viasuitable deuterated precursors according to the syntheses described,e.g., in U.S. Pat. No. 2,556,845, in the case of 4-vinylpyridine, or inTetrahedron Letters 1993, 8329 and in Journal of the American ChemicalSociety 75, 1953, 4738, in he case of 3-vinylpyridine.

FIG. 5 gives an example of this type of analysis. Sera from normal ratswere separately marked with “light” and “heavy” (hepta-deuterated)2-vinylpyridine, mixed in a 70% deuterated/30% light label, andseparated by 2-D mapping. The zone of the α₁-acid glycoprotein (M_(r)21546, pI 5.0) was eluted, digested with trypsin and analysed by massspectrometry in the MALDI-TOF mode. This analysis gave the mass spectrumof FIG. 5. A number of peptide fragments is observed at various m/zvalues, including the two fragments at m/z 1235.0 and 1241.9, Thedifference in m/z of 6.9 coincides with the difference between the2-d₇-VP and the non-deuterated 2-VP. This observation, together with theratio of the relative intensities of the two signals (ca. 68:32)confirms that this peptide contains a single Cys residue which reflectsthe ratio of the two isotopic markers (70:30) in the original proteinmixture.

1. A process for the selective alkylation of —SH groups in a protein,comprising the reaction in neutral or alkaline milieu of said proteinwith a weakly basic compound having: (a) one or more weakly basicnitrogen groups; (b) at least one acrylic-type double bond, wherein thepercent alkylation of the total —SH groups is higher than 90%.
 2. Aprocess according to claim 1, wherein the weakly basic compounds areselected from 2-vinylpyridine, 3-vinylpyridine or 4-vinylpyridine.
 3. Aprocess according to claim 2, wherein the weakly basic compounds areselected from 2-vinylpyridine and 4-vinylpyridine.
 4. A processaccording to claim 2 or 3 wherein the vinylpyridine are deuterated,either partially or totally.
 5. A process according to any one of claimsfrom 1 to 4, wherein the percent alkylation of the total —SH groups ishigher than 95%
 6. A process according to claim 5, wherein the percentalkylation of the total —SH groups is about 100%.
 7. A method for theanalysis of proteins by means of electrophoretic or analyticaltechniques, comprising a preliminary alkylation of the —SH groups of theproteins to be analyzed by the process of claims 1-6.
 8. A methodaccording to claim 7 wherein said electrophoretic or analyticaltechniques are selected from: proteome analysis or the analysis ofcomplex protein/peptide mixtures, either under naive or denaturedconditions; electrophoretic two-dimensional maps, said maps comprising afirst dimension by isoelectric focusing (either conventional or inimmobilized pH gradients) followed by a second SDS-PAGE dimension.electrophoretic methods, either mono-, bi- or multi-dimensional, eitherin a free phase or on gels; capillary electrophoresis, either in freephase or in sieving liquid polymers, in presence of SDS, in theisoelectric focusing mode, in conventional capillaries, in micro- andnano-chips; a combination of the above methods with blotting and massspectrometry, either on-line or off-line; chromatographic separations,either mono-, bi or multi-dimensional; mixed-type separations,electrophoretic/chromatographic, either bi- or multi-dimensional;pre-fractionation procedures in proteome analysis, or in general inanalysis of complex protein/peptide mixtures, via electrophoretic orchromatographic procedures of any kind, either singly or in combination.